ascl1 and gsh1/2 control inhibitory and excitatory cell fate in spinal sensory interneurons

Ascl1 and Gsh1/2 control inhibitory and excitatory cellfate in spinal sensory interneurons

Rumiko Mizuguchi1,4, Sonja Kriks1,4, Ralf Cordes3, Achim Gossler3, Qiufu Ma2 & Martyn Goulding1

Sensory information from the periphery is integrated and transduced by excitatory and inhibitory interneurons in the dorsal spinal

cord. Recent studies have identified a number of postmitotic factors that control the generation of these sensory interneurons.

We show that Gsh1/2 and Ascl1 (Mash1), which are expressed in sensory interneuron progenitors, control the choice between

excitatory and inhibitory cell fates in the developing mouse spinal cord. During the early phase of neurogenesis, Gsh1/2 and

Ascl1 coordinately regulate the expression of Tlx3, which is a critical postmitotic determinant for dorsal glutamatergic sensory

interneurons. However, at later developmental times, Ascl1 controls the expression of Ptf1a in dILA progenitors to promote

inhibitory neuron differentiation while at the same time upregulating Notch signaling to ensure the proper generation of dILB

excitatory neurons. We propose that this switch in Ascl1 function enables the cogeneration of inhibitory and excitatory sensory

interneurons from a common pool of dorsal progenitors.

Interneurons in the dorsal horn have essential roles in processing andtransducing exteroceptive and proprioceptive sensory information1.These sensory inputs are carried by afferent fibers that terminate onsensory interneurons in the superficial laminae of the dorsal horn. Theprimary function of these excitatory and inhibitory interneurons is tointegrate and gate diverse types of sensory inputs, including nociceptivestimuli1,2. Consequently, alterations in the balance of the number and/or transmission properties of these excitatory and inhibitory inter-neurons are thought to be major contributing factors for chronicsensory neuropathies such as hyperalgesia and allodynia3,4.

In mice, neurons in the dorsal horn are generated in two phases: anearly phase (embryonic day (E) 10–11.5) that generates the relayneurons and sensory interneurons of the deep dorsal horn, followedby a second phase (E12–E13.5) in which the interneurons of thesubstantia gelatinosa (lamina II) and laminae III/IVare born5,6. Duringthe early phase of dorsal neurogenesis, dorsoventral patterning signalsspecify six types of dorsal neurons, which can be subdivided into twomajor classes: Class A (dI1–dI3) relay neurons, which are induced bybone morphogenetic protein (BMP) signaling; and Class B (dI4–dI6)neurons, which express the homeodomain transcription factor Lbx1and develop in a BMP-independent manner6–9. Two early-born ClassB cell types, dI4 and dI5 neurons, generate sensory interneurons thatpredominantly settle in the deep dorsal horn6,9. dI4 neurons differ-entiate as GABAergic inhibitory neurons that express the homeodo-main transcription factors Pax2 and Lhx1/5, whereas dI5 neuronsdevelop as glutamatergic neurons that express the homeodomaintranscription factors Tlx1/3 and Lmx1b.

Most of the interneurons that form the superficial dorsal horn(laminae II–IV) are late-born cells (dIL neurons)5,6,9. dIL cells comprise

excitatory and inhibitory cell types: GABAergic dILA neurons thatexpress Pax2 and Lhx1/5, and glutamatergic dILB neurons that expressTlx1/3 and Lmx1b (refs. 6,10). Whereas inhibitory and excitatoryneuron cell types, including dI4 and dI5 neurons, are typicallygenerated from distinct progenitor populations11–18, late-born dILneurons arise from a single dorsal domain, the dIL progenitordomain6,9,16,17. The mechanism that generates cell types with opposingneurotransmitter phenotypes from this single late progenitor domain isnot known, nor is it clear how it differs from the one that operates atearlier times when dI4 and dI5 neurons arise from distinct progenitorpopulations. Notably, the transcription factor profiles of early and latedorsal progenitors are similar, suggesting that a change in the geneticinteractions between these factors might underlie the altered mode ofexcitatory/inhibitory neuron generation in the late dorsal spinal cord.Tlx1 and Tlx3 function in a cell-autonomous manner to specify a

glutamatergic cell fate in postmitotic dorsal neurons10. Mice lackingTlx1 and Tlx3 show a near complete loss of glutamatergic neurons inthe dorsal horn, whereas overexpression of Tlx3 generates ectopicexcitatory neurons at the expense of inhibitory neurons. In Tlx1/3mutant mice, Pax2 and inhibitory neurotransmitter markers areupregulated in all Lbx1+ sensory interneurons, indicating that Tlx1/3repress the inhibitory differentiation program. Pax2 also regulates theneurotransmitter phenotype of dorsal sensory neurons, insofar as it isrequired for these cells to differentiate as inhibitory neurons10. Pax2seems to function downstream of Ptf1a, a bHLH transcription factorthat controls the development of inhibitory neurons in the dorsalspinal cord11.

In this study, we show that genetic interactions involving Gsh1/2 andAscl1 in dorsal progenitors restrict the expression of Ptf1a and the

Received 14 March; accepted 25 April; published online 21 May 2006; doi:10.1038/nn1706

1Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037, USA. 2Department of CancerBiology, Dana Farber Cancer Institute, 1 Jimmy Fund Way, Boston, Massachusetts 02115, USA. 3Institute for Molecular Biology, Medizinische Hochschule Hannover, CarlNeuberg Strasse 1, Hannover, Germany. 4These authors contributed equally to the work. Correspondence should be addressed to M.G. ([email protected]).

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postmitotic determination factors Tlx1/3 andPax2 to different subsets of differentiatingsensory neurons, thereby specifying the iden-tity and neurotransmitter phenotype of dorsalsensory interneurons. Notably, these inter-actions change between the early and latephases of neurogenesis. Whereas Ascl1 func-tions downstream of Gsh1/2 to activate Tlx3 indI5 neurons in the early phase of dorsalneurogenesis, at later times it functionallyantagonizes Gsh1/2 by upregulating Ptf1aexpression in prospective dILA neurons. Thisupregulation of Ptf1a by Ascl1 is necessary forprospective dILA cells to differentiate as inhi-bitory neurons11. During the late phase ofneurogenesis, Ascl1 also modulates Notch sig-naling in adjacent dIL progenitors to promotea dILB fate, thereby reinforcing the switchbetween inhibitory and excitatory cell fates.We propose that this change in Ascl1 function,which involves the Ascl1-dependent recruit-ment of Notch signaling, enables excitatoryand inhibitory sensory neurons to developfrom a common pool of late dorsal precursors.

RESULTS

Dorsal progenitor gene expression

We compared the expression of Gsh1/2, Ascl1(Mash1) and Ptf1a in early and late dorsalprogenitors in sections from E10.5–E12.5spinal cords. In the early phase of neurogen-esis, Gsh1/2 and Ascl1 were coexpressedthroughout the dI3–dI5 progenitor domain(Fig. 1a–e)12,15. Ptf1a expression was morelimited, being restricted to dI4 progenitors(Fig. 1c,d,f,g)11. During the late phase ofneurogenesis, Gsh1/2 and Ascl1 expressionlargely overlapped in dIL progenitors(Fig. 1h), whereas Ptf1a showed a more mosaic distribution in dILprogenitors (Fig. 1i–k). Whereas Ascl1+/Ptf1a+ double-labeled cellswere present throughout the dIL domain (arrowheads in Fig. 1j), mostAscl1+ cells were located in the ventricular zone proper, whereas thePtf1a+ cells were present predominantly in the subventricular zone.These Ptf1a+ cells were, for the most part, Gsh1/2-negative (Fig. 1k,l).Many Ptf1a cells labeled with an antibody to Ki67 (SupplementaryFig. 1 online). After short (1–2 h) bromodeoxyuridine (BrdU) pulses,we occasionally observed double-labeled Ptf1a+/BrdU+ cells. In con-trast, we frequently observed Ascl1+ cells labeled with BrdU (data notshown). Ascl1 therefore seems to preferentially mark early dividing dILprogenitors, whereas Ptf1a is likely to be expressed in a subset of dILprogenitors that are exiting the cell cycle.

Gsh1/2 and Ascl1 specify sensory interneuron fate

Gsh1/2 and Ascl1 are required for the differentiation of early-bornexcitatory dI3 and dI5 neurons (Fig. 2a–d; data not shown; andrefs. 12,15). At this early time, Gsh1/2 functions upstream of Ascl1,where it is required for normal Ascl1 expression in dI3 and dI5progenitors (ref. 12 and data not shown). Gsh1/2 and Ascl1 weredispensable for the development of inhibitory dI4 neurons (Fig. 2a,c),and Ascl1 when overexpressed in the chick neural tube repressed Pax2in the domain in which dI4 neurons are normally located (Fig. 2e,f;

arrow in Fig. 2f). Ascl1 does, however, seem to be sufficient to specifydI5 neurons, insofar asAscl1 overexpression ectopically induced Lmx1b,a marker of dI5 neurons (Fig. 2g,h and ref. 15). Ascl1 also induced Tlx3(data not shown), a known determinant of dorsal glutamatergiccell fate10. Ascl1 is therefore able to induce dorsal glutamatergicinterneurons at the expense of dI4 inhibitory interneurons.

As Gsh1/2 and Ascl1 have crucial roles in specifying early-bornexcitatory dI5 neurons, we asked whether these genes also regulatethe neurotransmitter phenotype of late-born dIL neurons. We analyzedthe expression of a number of neurotransmitter-specific vesiculartransporter genes, including Slc32a1 (VIAAT; ref. 19) and Slc17a6(VGlut2; ref. 20) in the E14.5 spinal cord. E14.5 Gsh1/2–/–spinalcords showed a strong increase in Slc32a1 expression in the dorsalhorn (Fig. 3a,b), which was accompanied by a marked reduction inSlc17a6 expression (Fig. 3c,d). Few, if any, Slc17a6+ cells remained inthe superficial dorsal horn (arrow in Fig. 3d). There was, therefore, aloss of excitatory sensory interneurons coupled with an increase inneurons expressing inhibitory markers such as Slc32a1 in the superficialdorsal horn of the Gsh1/2–/– mutants.

E14.5 Ascl1–/– spinal cords showed the opposite phenotype: namely,a marked reduction in Slc32a1 expression (Fig. 3e,f). These changes inSlc32a1 expression were largely restricted to the superficial dorsal hornwhere dIL neurons are located. Gad1, Gad2 and Slc6a5 (GlyT2)

Gsh1/2+

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Figure 1 Expression of Gsh1/2, Ascl1 (Mash1) and Ptf1a in the dorsal spinal cord.

(a–k) Immunostainings of Gsh1/2, Ascl1 and Ptf1a in E10.5–E12.5 wild-type mouse embryos. At

E10.5, Gsh1/2 (red) and Ascl11 (green) were expressed in dividing dI3–dI5 precursors (a,b). Ptf1a (red)

was expressed in dI4 progenitors. Ascl1 (green) was expressed in dI3, dI4 and dI5 progenitors, albeit at

a reduced intensity in Ptf1a+ dI4 progenitors (c,d). At E11.5, Ascl1 (green) was coexpressed with Gsh2

(red) in dI3–dI5 progenitors, with the dI4 and dI5 progenitor domains giving rise to Lbx1+ (blue)

interneurons (e). At this stage, the Ptf1a expression domain began to expand, with most Ptf1a+ cells

expressing some levels of Ascl1 (f,g). At E12.5, late-born Lbx1+ neurons arose from a single progenitor

domain where Gsh1/2 and Ascl1 are coexpressed (h). At this stage, Ptf1a+ cells (red) were located

primarily at the lateral edge of the ventricular zone and expressed little or no Gsh1/2 (green) (i–k).

Ascl1 and Ptf1a were coexpressed in some dIL progenitors (j, arrowheads). (l) Schematic summary

showing the expression of Gsh1/2, Ascl1 and Ptf1a in early-born (E10–E11.5) and late-born

(E12.0–E13.5) sensory interneuron precursors. Lbx1+ dI4 and dILA neurons expressed Pax2

and differentiated as inhibitory (‘inh.’) neurons. Lbx1+ dI5 and dILB cells expressed Tlx1/3 and

differentiated as excitatory (‘exc.’) neurons. Bar: 50 mm in a, c, e, f, h, i, k; 20 mm in b, d, g, j.

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expression levels were also substantially reduced (data not shown),demonstrating that there is a general reduction of inhibitory neuro-transmitter gene expression in Ascl1–/– dIL neurons. An associatedincrease in Slc17a6 expression was visible in the dorsal horn, althoughmany of these Slc17a6+ cells were positioned medially, suggesting adefect in their migration (arrow in Fig. 3g,h). These findings, inaddition to providing further evidence that late-born excitatory andinhibitory neurons are derived from a common precursor pool,demonstrate that Ascl1 and Gsh1/2 have opposing roles in determiningthe neurotransmitter phenotype of late-born sensory interneurons.

Pax2 and Lmx1b expression in Gsh1/2 and Ascl1 mutants

To further investigate the nature of these changes to neurotransmittergene expression in dIL neurons, we analyzed the expression of Pax2 andLmx1b at E14.5. At this stage, the superficial dorsal horn is largelycomposed of Pax2+ dILA cells that are predominantly GABAergic andLmx1b+ dILB cells that are exclusively glutamatergic. Spinal cords fromE14.5 Gsh1/2 mutant mice showed a strong increase in the number ofPax2+ cells in the superficial dorsal horn (Fig. 3i,j; 420 ± 46 (s.d.) wild-type versus 748 ± 103 mutant cells per section), which is consistent withthe observed increase in Slc32a1 expression (Fig. 3a,b). Moreover,Lmx1b-expressing cells were completely missing from the dorsal horn(Fig. 3k,l; 471 ± 29 wild-type versus 4 ± 2 mutant cells per section),thereby accounting for the virtual absence of Slc17a6 expression in thesuperficial dorsal horn at E14.5.

E14.5 Ascl1–/– spinal cords showed the opposite phenotype: namely areduction in dorsal Pax2+ cells (Fig. 3m,n; 397 ± 38 wild-type versus

131 ± 27 mutant cells per section), coupled with an increase in Lmx1b+

cells (Fig. 3o,p; 466 ± 44 wild-type versus 828 ± 53 mutant cells persection). Ascl1 is therefore required for the proper development of late-born Pax2+ inhibitory neurons but is dispensable for the developmentof excitatory neurons. This contrasts with the finding that Ascl1 isrequired for the development of early-born excitatory dI5 neurons(Fig. 2d). Taken together, these results demonstrate that Gsh1/2 andAscl1 have opposing roles in determining the identity of the late-bornsensory interneurons that form the dorsal horn.

Ascl1 and Gsh1/2 control dIL cell fate

We then set out to determine if Gsh1/2 and Ascl1 function upstream ofTlx1/3 and Pax2 to regulate the fate of late-born dorsal progenitors. AtE12.5, newborn Lbx1+ dIL neurons are in transit through the subven-tricular zone where they begin expressing either Pax2 or Tlx (refs.6,9,10,21). Although late-born Lbx1+ dIL neurons were generated innormal numbers in the E12.5 Gsh1/2–/– mutant cord (Fig. 4a,b), thenumber of Pax2-expressing neurons was greatly increased (Fig. 4c,d;241 ± 23 wild-type versus 432 ± 28 mutant cells per section). Tlx3-expressing cells were completely absent from the Gsh1/2–/– dorsal horn(Fig. 4e,f; 301 ± 32 wild-type versus 2 ± 1 mutant cells per section),indicating that presumptive Lbx1+/Tlx3+ dILB neurons differentiate asLbx1+/Pax2+ dILA neurons. This loss of late-born Tlx3+ dILB neuronsparalleled the loss of early-born Tlx3+/Lmx1b+ dI5 neurons in theGsh1/2 mutant cord (Fig. 2b and ref. 12) and demonstrates that Gsh1/2control the generation of both early-born and late-born Tlx3+ gluta-matergic neurons.

E11.5 Gsh1/2 mutant E11.5 Ascl1 mutant

Ascl1 EP

Pax2

Pax2 GFP Pax2 Lmx1b GFP Lmx1b

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Figure 2 Gsh1/2 and Ascl1 are necessary for the specification of early-born

dI5 neurons. (a–d) Analysis of early-born neurons in Gsh1/2–/– and Ascl1–/–

mutants. In E11.5 dorsal spinal cords, Pax2 was expressed in dI4 and dI6

neurons but not in d5 neurons (a,c). In Gsh1/2–/– mice, cells in the dI5

domain expressed Pax2+ (arrow in a), indicating that these neurons had

adopted a dI4 or dI6 fate. Lmx1b+ dI5 neurons were essentially absent in

the Gsh1/2–/– spinal cord (arrow in b). A similar phenotype was observed

in Ascl1–/– mutants, with an expansion of Pax2+ dI4–dI6 neurons and acomplementary loss of Lmx1b-expressing dI5 neurons (arrows in c,d).

(e–h) Ascl1 and GFP expression vectors were coelectroporated into

E3 chick spinal cords and analyzed at E5. Overexpression of Ascl1

reduced the number of Pax2+ dI4 neurons (arrow in f) and increased

the number of Lmx1b+ cells adjacent to the dI5 domain (arrow in h).

Bar, 50 mm.

Figure 3 Switch in cell fate of late-born neurons in Gsh1/2–/– and Ascl1–/–

mice. (a–h) The dorsal horn of E14.5 Gsh1/2–/– and Ascl1–/– mice was

analyzed by in situ hybridization with Slc32a1 (VIAAT) and Slc17a6 (VGlut2)

as markers of inhibitory and excitatory neurons, respectively. Mice lacking

Gsh1 and Gsh2 showed an increase in the expression of the inhibitory marker

Slc32a1 in the substantia gelatinosa (arrow in a,b) with a concomitant

decrease in the expression of the excitatory marker Slc17a6 (arrow in c,d).

Ascl1–/– mice showed a complementary phenotype: the number of Slc32a1+

neurons was reduced (arrow in e,f), whereas the number of Slc17a6+ neurons

was increased (arrow in g,h). Many Slc17a6+ cells were observed medially

(arrow in h), suggesting that the migration of those neurons was also affected.

(i–p) Changes in the expression of Pax2 and Lmx1b in the superficial dorsalhorn of E14.5 Gsh1/2–/– and Ascl1–/– embryos. The number of dILA neurons,

marked by Pax2 expression, was increased in Gsh1/2–/– cords (i,j) whereas

Lmx1b neurons were almost completely absent (k,l), suggesting a switch from

dILB to dILA neuron identity. Ascl1–/– cords showed a marked reduction in

Pax2 expression, with a concomitant increase in the number of Lmx1b-

expressing cells (m–p). Bar, 50 mm.


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Although Lbx1+ dIL neurons were still generated, albeit in slightlyreduced numbers, in the Ascl1–/– cord (Fig. 4g,h), we noted an 80%reduction (202 ± 21 wild-type versus 34 ± 7 mutant cells per section) inthe number of late-born Pax2+ cells in the subventricular zone wherenewborn dILA neurons are normally located (circle in Fig. 4i,j). Incontrast, the number of Tlx3+ cells within the subventricular zone wasincreased (circle in Fig. 4k,l; 206 ± 8 wild-type versus 283 ± 26 mutantcells per section), demonstrating that dIL neurons preferentiallydifferentiate as dILB neurons in the Ascl1–/– cord. Whereas approxi-mately 50% of the Lbx1+ cells in the dorsal subventricular zone of E12.5wild-type spinal cords expressed Pax2+ (Fig. 4m), less than 10% of theLbx1+ cells in the subventricular zone of the Ascl1–/– spinal cord werePax2+ (Fig. 4n). Instead, most of these Lbx1+ cells expressed Lmx1b(Fig. 4o). This shows that Gsh1/2 and Ascl1 function in a geneticallyantagonistic manner to regulate the choice between dILA and dILB

identity; moreover, this finding demonstrates an important change inthe interactions that govern the differentiation of late-born sensoryneurons. Whereas both Gsh1/2 and Ascl1 were necessary for thedevelopment of early-born Tlx3+/Lmx1b+ excitatory neurons(Fig. 2), at later times Ascl1 was dispensable for Tlx3/Lmx1b expressionand excitatory neuron differentiation. Ascl1 was instead required forthe proper specification of Pax2+ dILA inhibitory neurons.

Altered Ptf1a expression in Ascl1 and Gsh1/2 mutants

Ptf1a is known to specify inhibitory neurons in both the cerebellumand the dorsal spinal cord11,22, raising the possibility that Ascl1 controlslate-born inhibitory differentiation by regulating Ptf1a expression in

presumptive dILA progenitors. At E10.5–E11.5, Ptf1a was still expressed in theAscl1–/– dI4 progenitor domain (Fig. 5a–d),which is consistent with the generation of dI4neurons at this stage. However, at E12.5 wedetected a marked reduction in Ptf1a expres-sion (Fig. 5e,f), which coincided with thereduced production of late-born dorsalPax2+ neurons (Fig. 4). Ascl1 is thereforerequired for the induction and/or mainte-nance of Ptf1a during the late, but not early,phase of dorsal sensory interneuron develop-ment. Gsh1/2 expression was unchanged inthe E12.5 Ascl1 mutant cord (Fig. 5g,h),demonstrating that Gsh1/2+ dIL progenitorspreferentially differentiate as excitatorydILB neurons when Ascl1 and Ptf1a are nolonger present.

The observation that Gsh1/2+ dorsal pro-genitors are biased toward becoming gluta-matergic neurons when Ascl1 is absentsuggested that the primary role of Ascl1 indIL progenitors might be to antagonize thefunction of Gsh1/2. We therefore looked to seeif Ascl1 and Ptf1a might be dispensable forinhibitory neuron differentiation when Gsh1/2are also absent. Although Ptf1a is expressed indI4 progenitors at E10.5 in the Gsh1/2–/– cord(Fig. 5i,j), E11.5 and E12.5 Gsh1/2–/– cordscontained fewer Ptf1a+ cells and reducedlevels of Ascl1 (Fig. 5k–p), even though thenumber of Pax2+ dILA neurons in these cordswas greatly increased (Fig. 4). Ascl1 and Ptf1aare therefore dispensable for dILB inhibitory

neuron specification in the dorsal spinal cord when Gsh1/2 areinactivated. The concomitant downregulation of Ascl1 and Ptf1a inthe Gsh1/2–/– cord also provides further evidence that Ascl1 regulatesPtf1a expression in dIL progenitors. These findings support a model inwhich Ascl1 regulates Ptf1a expression in a subset of dIL precursors,thus antagonizing the function of Gsh1/2 (model in SupplementaryFig. 2 online). In this model, Ascl1, by activating Ptf1a in prospectivedILA precursors, ensures that a subset of Gsh1/2+ dIL progenitorsdifferentiate as inhibitory neurons.

Ptf1a and Ascl1 suppress the dILB differentiation program

To test whether Ptf1a can block Tlx3 expression and dILB cell differ-entiation, we overexpressed Ptf1a in the E5 chick spinal cord when late-born neurons were being generated. Expression of Ptf1a caused amarked reduction in the number of Lmx1b+ cells in the dIL domain onthe electroporated side of the cord, demonstrating that Ptf1a blocks thedifferentiation of dILB excitatory neurons (Fig. 6a–d, arrow in b). Pax2and Lhx1/5 expression were largely unaffected (Fig. 6e,f). Ptf1a alsosuppressed Tlx3, an obligatory determinant of dorsal excitatory cell fate(Fig. 6d,g), a result that is consistent with the increase in Tlx3expression in the Ptf1a–/– spinal cord that accompanies the switchfrom dILA to dILB fate in these mutants11. We also noted an increase inthe density of Pax2+ and Lhx1/5+ neurons in the subventricular zoneon the electroporated side of the neural tube (arrows in Fig. 6e,f),further supporting a role for Ptf1a in promoting an inhibitory dILA fateover an excitatory dILB fate. Notably, Cash1 mRNA levels were reducedafter Ptf1a overexpression (Fig. 6h), which may explain the low

Tlx

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Figure 4 Changes in transcription factor expression in Gsh1/2–/– and Ascl1–/– mice at E12.5.

(a–l) Immunostainings for late-born neuron markers in the dorsal horn of E12.5 Gsh1/2–/– (a–f) and

Ascl1–/– (g–l) embryos. Lbx1, which is expressed in both dILA and dILB neurons, was largely unchanged

in the Gsh1/2–/– cord (a,b). Pax2+ dILA neurons were increased in the dorsal horn of the Gsh1/2–/–

mutant (arrow in c,d), whereas Tlx3+ neurons were completely lost in the Gsh1/2–/– mutant (e,f). In the

Ascl1–/– cord, Lbx1-expressing neurons, were slightly decreased in number (g,h). These embryos showed

a marked decrease in Pax2+ cells in the subventricular zone (svz, circled in i,j). Neurons expressing Pax2were still present in the more lateral region of the dorsal horn (j, arrow). These cells represented dI4

neurons and the dILA neurons born at E11.5 that had migrated laterally. Tlx3 was upregulated in the

Ascl1–/– cord in areas where Pax2 was decreased (circled in k,l). (m–o) In wild-type embryos,

approximately half of the Lbx1+ cells (green) in the subventricular zone expressed Pax2 (red) (m). In

contrast, Ascl1–/– embryos had very few Lbx1/Pax2 double-labeled neurons in the subventricular zone

of the dorsal horn (n); rather, most cells in this zone coexpressed Lbx1 (green) and Lmx1b (red) (o).

Bar: 50 mm in a–l; 25 mm in m–o.


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expression of Ascl1 in many cells with high level expression of Ptf1a(Ptf1ahigh) in the dIL subventricular zone (Fig. 1i,j).

When Ascl1 was misexpressed in late progenitors, we saw amarked reduction in Lmx1b, a marker of dILB excitatory neurons(Fig. 6i–l, arrows in j and k). This led us to test whether Ascl1 mightrepress dILB differentiation by inducing dILA cell fates. Althoughwe observed an upregulation of Ptf1a mRNA in the electroporatedhalf of the cord (Fig. 6m, arrows) after electroporating Ascl1, therewas no obvious induction of either Pax2 or Lhx1/5 (data not shown).The failure to see an increase in the number of dILA cells at this stagemay be related to the neurogenic activity of Ascl1, which has beenshown to promote precocious neuronal differentiation and depletethe progenitor pool. To examine more carefully whether Ascl1 canpromote a dILA fate, E6 spinal cords were electroporated with an Ascl1expression vector and analyzed 24 h later. Under these circumstances,we saw some precocious induction of Pax2 in the ventricular zone onthe electroporated side of the cord (Fig. 6n–p, arrowheads in o).Many of these Pax2+ cells were located medially in the ventricularzone (Fig. 6o), and in all instances these cells expressed Ascl1

(arrowheads in Fig. 6p), suggesting that Ascl1 can induce Pax2 in acell-autonomous manner.

Notch signaling regulates the fate of dIL progenitors

Ascl1 is known to act cell autonomously to promote neuronaldifferentiation and specification23; however, Ascl1 also upregulatesthe Delta (Dll) genes24, which in turn activate Notch signaling insurrounding cells. Thus in principle, Ascl1 could either actautonomously or non–cell autonomously to promote an inhibitorydILA cell fate. In examining Ascl1 and activated Notch (NICD)expression in the subventricular zone at E12.5, we observed alargely complementary pattern of expression: NICD was low in cellswith an elevated expression of Ascl1 (arrows in Fig. 7a–d) and viceversa (arrowheads). Moreover, in Ascl1 mutant cords, NICD levelswere reduced (Fig. 7e,f), demonstrating that Ascl1 is required toactivate Notch signaling in these surrounding cells. We also observedmarkedly reduced Dll1 levels in dIL progenitors in the Ascl1 mutantcord at E12.5 (arrow in Fig. 7g,h), demonstrating that Dll1 is down-stream of Ascl1. These findings imply that Ascl1 not only acts cell

autonomously to induce dILA fate, but mayalso function non–cell autonomously to sup-press dILA fate in adjacent cells by activatingNotch signaling in them.

When Delta is expressed broadly, itblocks Notch signaling in a cell-autonomousmanner23, whereas mosaic Delta expressionactivates Notch signaling in adjacent cells.To test whether Notch signaling is involvedin specifying late-born neurons, we per-formed a series of Dll1 misexpression experi-ments in chick embryos. Chick E5 embryoswere electroporated with various amountsof a Dll1 expression vector and analyzed fordIL subtype markers (Fig. 8a–h andSupplementary Fig. 3 online). When Dll1was introduced at low levels that producedmosaic expression in dIL progenitors (Fig. 8a),a marked upregulation of Tlx3 was seen(arrowheads in Fig. 8d), with increased num-bers of Tlx3+ cells in the dIL domain on theelectroporated side versus the unelectro-porated side (89 ± 16 versus 59 ± 9 cells persection; n ¼ 4 sections; 4 electroporatedembryos). Few, if any, Tlx3+ cells were Dll1+,which is consistent with Dll1 inducing dILB

differentiation in a non–cell autonomousmanner. When a vector containing the GFPreporter alone was electroporated into theneural tube, the number of cells on the elec-troporated side was unchanged compared tothe unelectroporated side (58 ± 12 versus 62 ±8 cells per section; n ¼ 4 sections; 2 electro-porated embryos). In contrast, when Dll1 wasmore broadly expressed in dIL progenitors, wesaw a substantial reduction in the number ofLmx1b+ dILB neurons (arrow in Fig. 8g) andthe few remaining Lmx1b+ cells on the elec-troporated side did not coexpress Dll1 (GFP)(arrowheads in Fig. 8h), suggesting that therepression of Lmx1b may occur in a cell-autonomous manner. Notably, we saw only

Ascl1Gsh1/2

E12

.5E

12.5

E11

.5E

10.5

Ptf1a Ptf1a

Gsh1/2 mutantAscl1 mutant

wt –/– wt –/–

wt –/– wt –/–

wt –/– wt –/–

wt –/– wt –/–

a b i j

c d k l

e f m n

g h o p

Figure 5 Altered Ptf1a expression in Gsh1/2–/– and Ascl1–/– mice. (a–n) Expression of Ptf1a in Ascl1–/–

(a–f) and Gsh1/2–/– (i–n) spinal cords. Ptf1a expression was strongly reduced at E12.5 (e,f), but not at

E10.5 and E11.5 (a–d), in the ventricular zone (vz) of Ascl1–/– cords relative to age-matched wild-type

(‘wt’) cords. This decrease was not due to a change in Gsh1/2 expression, as Gsh1/2 showed a normal

expression pattern at E12.5 in Ascl1–/– embryos (g,h). Expression of Ptf1a was also markedly decreased

at E11.5 and E12.5 (k–n), but not at E10.5 (i,j), in the Gsh1/2–/– cord. Ascl1 was strongly reduced in

the Gsh1/2–/– cord at E12.5 (o,p). Bar, 50 mm.


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minor changes in the number of dorsal Pax2+ cells following high Dll1misexpression (Fig. 8b,f). It therefore seems that the activation ofNotch signaling in dIL progenitors biases them toward a dILB fate, butis not necessary in order for them to differentiate as dILA neurons.

We then analyzed Psen1 (Presenilin-1) mutants that have reducedlevels of Notch signaling24,25 to test whether Notch signaling is indeednecessary for generating dILB neurons and dispensable for dILA

development (Fig. 8i–l). First, we confirmed that the level of activatedNotch in Psen1–/– dIL progenitors is greatly reduced compared to thatin their wild-type counterparts (Fig. 8i). In keeping with previousfindings that Notch signaling suppresses Ascl1 expression26,27, we alsoobserved a sharp increase in the density of Ascl1high cells in the dorsalventricular zone of the Psen1–/– mutant at E12.5 (Fig. 8j). This loss of

Notch signaling in dIL progenitors in thePsen1–/– cord was associated with a reductionin Lmx1b+ cells in the dorsal subventricularzone, where dILB neurons are normally gen-erated (dashed circles in Fig. 8k,l). At bothE12.5 (data not shown) and E14.5 (Supple-mentary Fig. 4 online), there was very little

change in the number of Pax2 cells, although the ratio of Pax2+ dILA

cells to Lmx1b+ dILB cells was increased (Supplementary Fig. 4). Forinstance, E14.5 Psen1–/– cords contained similar numbers of Pax2+ cellsin the dorsal horn (781 ± 46 wild-type versus 748 ± 51 mutant cells persection), whereas the number of dILB neurons in the Psen1–/– dorsalhorn was reduced by 440% (814 ± 43 wild-type versus 472 ± 38mutant cells per section; Supplementary Fig. 4), indicating that Notchsignaling is required to generate a normal complement of dILB

neurons. The ratio of Ptf1a+ cells to Pax7+ dorsal progenitors wasincreased in the E12.5 Psen1–/– cord (Supplementary Fig. 4;

Ascl1 Pax2Ascl1 Pax2 Pax2Ptf1a (mRNA)

Cash1 (mRNA)

GFP Lmx1b Lmx1bAscl1 EP

Pax2 Lhx1/5 Tlx3

Lmx1b GFPPtf1a EPLmx1b

EP

*E

P*

EP

*E

P*

LateEarlyLmx1b+

Lmx1b+ Tlx3+

0

50

100

Cel

l num

ber

0

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100

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l num

ber

*

*

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e f g h

i j k l

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Dll1NICD Lbx1

NICD Ascl1 NICD

Ascl1 mutant

wt –/–

Ascl1

wt –/–

* * *

a b c d

e f g h

Figure 6 Ptf1a and Ascl1 overexpression

represses the dILB fate of late-born interneurons.

(a–h) Chick spinal cords were electroporated with

a Ptf1a-IRES-EGFP expression vector at E5 and

analyzed at E7 for the staining of the indicated

markers (red) and GFP (green). Lmx1b+ dILB

neurons were strongly repressed following

electroporation with Ptf1a (a–d). Arrow in b pointsto reduced numbers of Lmx1b+ neurons on the

electroporated side. c shows a higher

magnification of b. Pax2 and Lhx1/5, markers

of dILA neurons, were induced following Ptf1a

overexpression (arrows in e,f), whereas Tlx3 was

markedly reduced on the electroporated side of

the spinal cord (g). Endogenous Cash1 mRNA

was also markedly reduced following over-

expression of Ptf1a (h). (i–l) E5 chick spinal

cords were coelectroporated with Ascl1 and GFP

expression vectors and similarly analyzed at E7.

Ascl1 repressed the generation of Lmx1b+ dILB

neurons (arrows in i–k), whereas the number of

early-born Lmx1b+ dI5 neurons was unchanged

(j, asterisk). k is a higher magnification of j.

Ptf1a mRNA was highly upregulated following

electroporation with Ascl1 (m). (n–p) E6 spinal

cords were electroporated with an Ascl1-ER

expression vector and analyzed 24 h later. At thisstage, Ascl1-expressing cells (green) were still

located in the ventricular or subventricular zones.

Induction of Pax2 (red) in cells expressing Ascl1

(green) was seen in the dorsal ventricular zone

(arrowheads in o,p). Bar, 50 mm.

Figure 7 Altered Notch signaling in Ascl1 mutants. (a–d) Ascl1 (green) and

activated Notch (NICD) (red) were expressed in a complementary pattern in

dorsal dIL progenitors at E12.5. Most of the Ascl1high cells expressed low

levels of NICD (arrows in b–d), whereas many of the cells with high

expression of NICD (NICDhigh) expressed low levels of Ascl1 (arrowheads

in b–d). A few cells coexpressed both proteins at an intermediate level

(asterisk). b–d show a higher magnification of a. (e–h) Expression patterns of

NICD and Dll1 were analyzed in wild-type and Ascl1–/– cord at E12.5. The

level of NICD (red) expression was strongly reduced in Ascl1–/– embryos

compared to wild-type littermates (e,f). Dll1 expression was also markedly

decreased in Ascl1–/– dorsal progenitors (arrows) at E12.5 (g,h). Bar: 50 mm

in a, e–h; 25 mm in b–d.


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0.10 wild-type versus 0.15 mutant), which is also consistent with theincreased ratio of Pax2+ dILA cells in the dorsal horn of these mutants.

We also analyzed the development of late-born dorsal interneuronsin Dll1 hypomorphic mutant embryos that show reduced levels ofNotch signaling (R.C. and A.G., unpublished data). Similar numbers ofPax2+ dILA cells were present in the wild-type and mutant cord (291 ± 30and 276 ± 21 cells per section, respectively; Fig. 8m,n). In contrast,the number of Lmx1b+ dILB cells was greatly reduced in the mutant(Fig. 8o,p; 286 ± 28 wild-type versus 187 ± 27 mutant cells per section).Consequently, the loss of Dll1 activity and Notch signaling leads to thepreferential loss of dILB neurons, providing further evidence that Notchsignaling, while dispensable for inhibitory Pax2+ neuron differentiation,is required for generating normal numbers of dILB neurons.

The demonstration that reduced Notch signaling favors the loss ofdILB cells, coupled with the expansion of the dILB population in theAscl1–/– cord (Fig. 4) where Notch signaling is attenuated (Fig. 7e,f),suggests that the primary function of Notch signaling is to limit theactivity of Ascl1 in a subset of dIL progenitors, thereby promoting anexcitatory dILB fate over an inhibitory dILA fate. We propose that Ascl1has two functions in dIL progenitors. First, it acts cell autonomously toinduce Ptf1a in dILA progenitors. Second, it upregulates Dll1 tosuppress the dILA program in nearby prospective dILB cells viaNotch-mediated lateral inhibition. Our findings are also consistentwith a model in which the cells expressing Ascl1 and high levels of Dll1(and thus low NICD) differentiate as dILA neurons, whereas dILprogenitors in which Notch signaling is high (and Dll1 is low) adopta dILB cell fate (Supplementary Fig. 2).

DISCUSSION

This study outlines the nature of the geneticinteractions at the precursor cell level thatcontrol the binary decision between excitatoryand inhibitory sensory interneuron fates in

the dorsal horn. In so doing, it reveals a critical role for Notch signalingin generating neuronal diversity within the developing vertebratenervous system. During the initial wave of dorsal neurogenesis, Ascl1and Gsh1/2 act in concert to specify excitatory sensory cell fates (ref. 12and this study). However, at later times when excitatory and inhibitoryneurons are cogenerated from a common pool of progenitors, Ascl1 isno longer needed for excitatory neuron specification. Instead, Ascl1regulates the expression of Ptf1a in a subset of dIL progenitors, therebydirecting them to differentiate as dILA inhibitory interneurons.Ascl1-dependent Notch signaling also has an integral role in this switchby promoting dILB cell fate over a dILA fate.

Specification of early-born sensory interneurons

During the early phase of neurogenesis, Gsh1/2 and Ascl1 functionsequentially to specify early-born dI5 neurons that differentiate asglutamatergic sensory interneurons. Ascl1 positively regulates dI5neuron development by instructively promoting the expression ofTlx3 and Lmx1b (Fig. 2) in these cells, thereby predisposing them toan excitatory fate. Gsh1/2 functions to maintain Ascl1 expression withinthe dI3–dI5 progenitor domains, in part by repressing Ngn1 (Neurog1)(ref. 12; and S.K., unpublished data).

A number of lines of evidence suggest that Gsh1/2 activity biasesdorsal progenitors to differentiate as excitatory neurons, which raisesthe question of why dI4 progenitors that express Gsh1/2 differentiate asinhibitory neurons. We propose that the production of inhibitoryneurons from Gsh1/2-expressing progenitors depends on a functionalblockade of Gsh1/2 by Ptf1a. In support of this model, Ptf1a expression

Lmx1b

Lmx1bAscl1NICD

Pax2 Lmx1b

Lmx1b GFP

Tlx3 GFPTlx3Pax2

Lmx1bPax2

Dll1 mutant

Psen1 mutant

Dll1 (rat)

wt –/– wt wt–/– –/–

wt –/–wt –/–

Dll1 EP (high)

Dll1 EP (low)Dll1 (rat)

a b c d

e f g h

i j k l

m n o p

Figure 8 Notch signaling regulates late-born

sensory interneuron specification. (a–h) E5 chick

spinal cords were electroporated with a Dll1-IRES-

EGFP expression vector and analyzed at E7. d and

h show higher magnification of electroporated

sides of c and g, respectively. Right and left

panels of d and h show the same area, with or

without GFP. When Dll1 was misexpressed at lowlevels (a–d), the expression of Tlx3 was induced

in a non–cell autonomous manner (c). Note that

very few ectopic Tlx3+ cell coexpressed GFP

(d, arrowheads). The expression of Pax2 on the

electroporated side was largely unchanged.

(b). When Dll1 was misexpressed at high levels

(e–h), which is known to reduce Notch activity,

expression of Lmx1b was strongly suppressed in

a cell-autonomous manner. The few Lmx1b+ cells

that remained (h, arrowheads) did not express

GFP. (i,j) E12.5 Psen1–/– cords showed reduced

NICD expression (i), which was accompanied by

an increase in the density of Ascl1high cells in

the dIL progenitor domain compared to that in

wild-type littermates (j). Note the reduced

dorsal progenitor domain in the Psen1–/– cord.

(k,l) Lmx1b+ dILB neurons were significantly

(P o 0.01) reduced in the subventricular zone

of the Psen1–/– cord (circles). (m–p) The E12.5Dll1hypo/– cord had fewer Lmx1b+ dILB neurons in

the subventricular zone (o,p, circles), whereas the

number of Pax2+ dILA neurons (red) was

unchanged (P 4 0.2; m,n). Bar: 50 mm.


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in the dI4 domain is regulated independently of Ascl1 at E10–E11.5(Fig. 5), and early-born Pax2+ dI4 neurons continue to be generatedfrom Gsh1/2+ progenitors in the Ascl1–/– cord (Fig. 2). Furthermore,we have found that Ptf1a can suppress Tlx3 (Fig. 6), which inturn functions as a repressor of inhibitory neuron development inthe dorsal spinal cord10. Our model predicts that Ptf1a is dispensablefor inhibitory neuron development, when Gsh1/2 are absent. Twolines of evidence support this. First, virtually all dIL-derivedLbx1+ cells adopt an inhibitory cell fate in the Gsh1/2–/– cordwhere Ptf1a expression is markedly reduced at E11.5 and E12.5(Fig. 5). Second, whereas Ptf1a is required for the differentiation ofinhibitory interneurons in the cerebellum and spinal cord11,22,other inhibitory neurons in the CNS develop in a Ptf1a-independentmanner. Consequently, Ptf1a is not an mandatory determinant ofinhibitory cell fate.

Specification of late-born sensory neurons

During the late phase of dorsal neurogenesis, there is a change in thegenetic interactions that generate excitatory and inhibitory sensoryinterneurons. The progenitors of these two classes of sensory inter-neurons seem to be bipotential, as dILA and dILB neurons are generatedin a ‘salt-and-pepper’ fashion from a single progenitor domain6,9 andinactivation of Gsh1/2 and Ascl1 produce largely compensatory shifts inthe numbers of dILA and dILB cells. Although we cannot exclude thepossibility that dILA and dILB precursors are distinguished by otherunidentified factors, their progeny can switch their identity and Ascl1clearly has a central role in regulating this switch.

During the late phase of neurogenesis, Ascl1 seems to exert its effectboth cell autonomously and non–cell autonomously. This change inAscl1 functionality is partly due to the Ascl1-dependent activation ofNotch signaling in late dorsal precursors. Whereas activated Notch(NICD) is very low in the early dorsal progenitors that generate dI4 anddI5 neurons (Supplementary Fig. 5 online), a subset of dIL progenitorcells express high levels of NICD, and this high expression of NICDdepends on Ascl1 (Fig. 7). Furthermore, NICD expression is highest incells that express low or no Ascl1 (Fig. 7), which suggests that Ascl1 isrequired to activate Notch in a non–cell autonomous manner. Wepropose that the coupling of Ascl1 to Notch signaling in dIL progeni-tors underpins the altered mode of excitatory and inhibitory neurongeneration in late dorsal precursors. The mechanism by which Ptf1aexpression and Notch signaling becomes dependent on Ascl1 in late(but not early) progenitors remains to be determined. Our data areconsistent with the presence of a factor in dI4 progenitors that controlsthe expression of Ptf1a in an Ascl1-independent manner. Consequently,the later dependence of Ptf1a expression on Ascl1 in late dorsalprogenitors might be due to the downregulation of this, as yetunidentified, factor. It is also possible that other factors may act inconcert with Ascl1 at later times to induce Ptf1a and Pax2 in dILA cells.

The misexpression of Dll1 in chick spinal cords, together with theanalysis of Psen1–/– and Dll1hypo/– mutants, demonstrates that attenu-ating Notch activity reduces the number of dILB neurons, therebysuggesting that Notch signaling normally promotes a dILB fate over adILA fate. Notch is also known to block Ptf1a function28 and so theAscl1-dependent activation of Notch in sibling dIL daughter cellswould lead to the repression of Ptf1a activity in presumptive dILB

progenitors, thereby blocking the dILA differentiation program in thesecells. Taken together, these findings clearly illustrate how the Ascl1-dependent activation of Notch provides a mechanism for generatinginhibitory and excitatory neurons from bipotential Gsh1/2+ progeni-tors. The model we propose (Supplementary Fig. 2) also predicts thatNotch signaling is not required for dILB differentiation when Ptf1a is

absent, which is precisely what occurs in the Ascl1–/– cord where Ptf1a isdownregulated (Fig. 5 and Fig. 7).

Relationship between early and late sensory interneurons

An important issue that arises from this study is the relationshipbetween the early-born Class B dI4 and dI5 neurons and their late-borndILA and dILB counterparts. dI4 and dILA neurons have a similartranscription factor profile, as do dI5 and dILB neurons6,9,10,16. Wepropose that early-born and late-born sensory interneurons are closelyrelated to each other, with the late-born dIL cells representing anexpansion of the early dI4 and dI5 populations. Notably, the dILinterneurons are generated at later developmental times when sensoryinterneuron progenitors are no longer segregated into distinct dorso-ventrally segregated populations6,9. This necessitates a mechanism thatgenerates excitatory and inhibitory cells types from a common pro-genitor pool. Our findings provide evidence that Ascl1 fulfills thisfunction by regulating Ptf1a expression and Notch signaling in dILprogenitors. In this way, Gsh1/2+ progenitors that are normally biasedtoward an excitatory fate give rise to a mixture of both inhibitory andexcitatory cell types.

This study provides one of the first detailed descriptions of howNotch signaling is integrated into a transcriptional switch that controlsthe choice between two different neuronal fates in the vertebratenervous system. In this instance, the recruitment of Notch is instru-mental in generating two populations of neurons with opposingneurotransmitter phenotypes from a single pool of progenitors.Many of the early classes of neurons in the developing spinal cordgive rise to multiple cell types8,29,30 and as such, Notch signaling mayprovide a common mechanism throughout the vertebrate nervoussystem for generating neuronal diversity.

METHODSAnimals. Gsh1 (ref. 31) and Gsh2 (ref. 32) heterozygous mice were obtained

from S. Potter and K. Campbell ( Children’s Medical Research Foundation,

Cincinnati), respectively. F. Guillemot (National Institute for Medical Research,

London) provided the Ascl1 (ref. 33) heterozygous mice. Psen1 heterozygous

mice were purchased from the Jackson Laboratory. The genotyping of mice has

been described previously12. Embryos were obtained from timed pregnancies,

with E0.5 being the detection date of the vaginal plug. All animal procedures

were undertaken according to the guidelines of the Institute Animal Care and

Use Committee at the Salk Institute.

Immunohistochemistry. Immunohistochemistry was performed as previously

described34. Antibodies to the following proteins were used: Gsh1/2 (rabbit

polyclonal), Gsh2 (rabbit polyclonal; K. Campbell), Ki67 (rabbit polyclonal),

Mash1 (mouse monoclonal/rabbit polyclonal; D. Anderson, Caltech, Pasade-

na), NICD (rabbit polyclonal), phospho-H3 (rabbit), Ptf1a (rabbit polyclonal;

H. Edlund, University of Umea, Sweden), Lbx1 (rabbit polyclonal and rat

polyclonal)34, Pax2 (rabbit polyclonal), Lhx 1/5 (mouse monoclonal 4F2-10),

Lmx1b (guinea-pig polyclonal, mouse monoclonal 50.5A5; T. Jessell, Columbia

University, New York) and Tlx3 (rabbit polyclonal; T. Muller. Max Delbruck

Centrum, Berlin). Species-specific secondary antibodies conjugated to Cy2, Cy3

and Cy5 were used to detect primary antibodies.

In ovo electroporation. The Ascl1 expression construct was described pre-

viously12. Full-length Ptf1a was amplified from E11.5 mice spinal cord cDNA

and cloned into the pCAGGS-IRES-EGFP and RCASBP expression vectors.

Full-length rat Dll1 cDNA was obtained from J. Boulter (University of

California, Los Angeles) and cloned into the pCAGGS-IRES-EGFP vector.

Electroporations were performed as previously described35. Embryos electro-

porated at E3, E5 or E6 were incubated for a further 24–48 h before being

processed for immunohistochemistry or in situ hybridization. GFP expression

was used to assess electroporation efficiency.


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In situ hybridization. In situ hybridizations were performed as previously

described36,37 using RNA probes for mouse Slc32a1 (ref. 19) and Slc17a6

(ref. 20) and a partial (492 bp) sequence of chick Ptf1a that was amplified from

E5 chick spinal cord cDNA. A full-length Cash1 (ref. 38) cDNA probe was

provided by M. Bronner-Fraser (Caltech, Pasadena).

Note: Supplementary information is available on the Nature Neuroscience website.

ACKNOWLEDGMENTSThe authors are grateful to S. Potter and K. Campbell for Gsh1 and Gsh2heterozygous mice. Ascl1 heterozygous mice were provided by F. Guillemot.We thank J. Johnson, M. Bronner-Fraser and J. Boulter for Ptf1a, Cash1 andDll1 cDNAs. Antibodies were provided by K. Campbell, T. Jessell, S. Morton,H. Edlund, T. Muller and D. Anderson. We also thank J. Johnson for sharingdata before submission. E. Lamar, C. Kintner, G. Lanuza and O. Britz providedparticularly helpful comments on the manuscript. This research was supportedby grants from the US National Institutes of Health to M.G. and Q.M.

AUTHOR CONTRIBUTIONSR.M. and S.J. undertook all the experiments for this study. R.C. and A.G.generated the Dll1hypo mice and embryos used in this study. M.G., Q.M.,R.M. and S.K. contributed to the experimental planning and writing ofthe manuscript.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Published online at http://www.nature.com/natureneuroscience

Reprints and permissions information is available online at http://npg.nature.com/

reprintsandpermissions/

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ascl1 and gsh1/2 control inhibitory and excitatory cell fate in spinal sensory interneurons

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